Effects of corneal preservation conditions on human corneal endothelial cell culture

The purpose of this study was to investigate the growth capacity of human corneal endothelial cells (HCEnCs) isolated from old donor corneas preserved in 4 different storage conditions. The following conditions were evaluated, A) cold storage (CS) (Optisol GS) for 7 days at 4 degrees C [n = 6]; B) organ culture (OC) (Cornea Max) for 7 days at 31 degrees C [n = 6]; C) OC for 28 days at 31 degrees C [n = 6] and; D) CS for 7 days at 4 degrees C followed by OC for 28 days at 31 degrees C [n = 6]. Following preservation, the Descemet membrane-endothelium complex was peeled and digested using Collagenase-Typel and was subsequently trypsinized before being plated into 2 wells (from each cornea) of an 8-well chamber slide. Media was refreshed every alternate day. The confluence rate (%) was assessed, and overall viability was determined using Hoechst, Ethidium Homodimer and CalceinAM staining. HCEnC-associated markers ZO-1, Na+/K+-ATPase, CD166 (Tag1A3), PRDX-6 (Tag2A12) and proliferative marker Ki-67 were used to analyse the cultures established from each condition. Donor tissues preserved in hypothermia (condition A) resulted in 9.3% +/- 4.0% trypan-blue positive cells (TBPCs) hence lower number of HCEnCs was plated. < 1% TBPCs were observed in conditions B, C and D. Indicatively, confluence in conditions A, B, C and D was 14.0%, 24.8%, 23.4% and 25.4% respectively (p = 0.9836) at day 1. By day 9, HCEnCs established from all conditions became confluent except cells from condition A (94.2% confluence). All HCEnCs in the 4 conditions were viable and expressed HCEnC-associated markers. In conclusion, OC system has advantages over hypothermic media for the preservation of older donor corneas rejected for corneal transplant and deemed suitable for corneal endothelial cell expansion, with lower TBPCs before peeling and longer period of tissue preservation over hypothermic storage system.